Cytoplasmic chaperones in precursor targeting to mitochondria: the role of MSF and hsp 70

Cytoplasmic chaperones in precursor targeting to mitochondria: the role of MSF and hsp70 Despite extensive study since the early 198Os, the mechanism by which newly synthesized protein precursors are unfolded in the cytoplasm and targeted correctly to the mitochondrial to translocation

through the mitochondrial

understood poorly. Recently, an N-ethylmaleimide cytopbsmic factor called mitochondrial

sur$ace prior membranes is

(iVEM)-sensitive

import stimulation

factor

(MSF), which catalyses the ATP-dependent unfolding ofprecursor proteins, was described. Unlike the more general chaperone proteins

of the hsp70 families, MSF not only unfolds proteins but also targets the unfolded precursor proteins to the mitochondria.

Here,

Mihara and Omura summarize what is known about MSF and speculate on how it, and other cytoplasmic factors, may be involved in mitochondrial

The authors are at the Dept of Molecular Biology, Graduate School of Medical Science, Fukuoka 812, Japan.

104

import.

Protein trafficking into eukaryotic organelles and across the prokaryotic plasma membrane is mediated by the interaction of discrete targeting signal sequences within precursor proteins, and involves specific components both in the cytosol and on the target membranes; in this way, precursor proteins are directed efficiently to specific membranes in an unfolded state suitable for translocation1-3. The processesinvolved have been analysed in depth in the case of protein translocation across the membrane of the endoplasmic reticulum (ER; Ref. 4). In this case, the signal recognition particle (SRI’) binds to the signal peptides of newly synthesized proteins as soon as they emerge from the ribosomes. The binding of SRP arrests the elongation of the peptide and targets the complex, composed of ribosome, nascent peptide and SRI’, to the SRPreceptor on the ER membrane. The formation of this complex maintains the translocation competence of the precursor by preventing folding of the protein4. 0 1996 Elsevier Science Ltd

For mitochondrial import, the mitochondrialtargeting sequences and the membrane components of the translocation machinery have been characterized extensively5. However, relatively little is known about how the mitochondrial signal sequences are specifically recognized and how the precursor proteins are targeted to the outer mitochondrial membrane6-ll. In the 198Os, several putative cytosolic components necessary for mitochondrial protein import were described (Table 1)12-14.For example, a component in a reticulocyte lysate was partially defined that interacts with mitochondria and is required for the specific binding to mitochondria of protein precursors14. Additionally, a stimulation factor with an apparent molecular mass of -40 kDa was partially purified from yeast cytos01~~.Later studies showed that some 70-kDa heat shock proteins (Ssalp, Ssa2p and hsp70) are involved in the import of proteins into mitochondria, as well as into the ER and nuclei. This suggested that hsp70 can function as a general factor that recognizes unfolded proteins at different intracellular locations and maintains them in an unfolded, import-competent state. However, the range of organelles into which import is stimulated by hsp70 suggests that hsp70 is not responsible itself for the specificity of targetinglGzO. Ydjlp (also known as MasSp), a homologue of Escherichia coli DnaJ has subsequently been identified as a cytoplasmic import factor possibly responsible for supplying the targeting function to hsp70 for import into mitochondria and the ER21-23.Ydjlp/MasSp is farnesylated and may act together with hsp70, probably at or near the membranes of these two organelles, to stimulate protein importz2. None of the factors described above totally satisfies all the criteria for a factor involved specifically in mitochondrial import. Most importantly, such a factor would be expected to recognize the mitochondrial-targeting sequence in the presequence. Two factors have been purified from rabbit reticulocyte lysates that fulfil this criterion, namely, ‘targeting factor’24,25and ‘presequence-binding factor” (PBF)26,and yet another cytosolic factor sensitive to N-ethylmaleimide (NEM) appears to be involved in yeast mitochondrial protein import. This latter factor, although it has not been purified, stimulates import in conjunction with Ssalp and Ssa2p, and may also play a role in precursor recognition and mitochondrial targeting17. Probably the most promising candidate studied to date is an NEM-sensitive factor that has been purified from rat liver cytosol. This factor, named mitochondrial import stimulation factor (MSF; Ref. 27), stimulates precursor import into mitochondria in vitro. Recent work has revealed that MSF is a unique chaperone protein that recognizes the mitochondrial-targeting sequences of precursor proteins and targets them to mitochondria, where they interact with the receptor on the mitochondrial outer membranezs31. All the results to date indicate that MSF is not a general molecular chaperone but, instead, a bona fide protein-import factor for mitochondria. Here, we briefly describe targeting factor and PBF, then summarize the function of both hsp70 and trendsin

CELL BIOLOGY

(Vol.

6) March

1996

TABLE

the unique chaperone protein, MSF, in the targeting of precursor proteins to mitochondria. Targeting

factor

and PBF

1 - POSSIBLE

MITOCHONDRIAL

Component

Source

Unpurified Partially purified Ssal p, Ssa2p NEM-sensitive facto? Targeting factor PBFb Ydjl p/Mastip MSF

Reticulocyte Yeast Yeast Yeast Reticulocyte Reticulocyte Yeast Rat liver

IMPORT

STIMULATION Mol.

mass

FACTORS (kDa)

-I

40

The in vitro import into mitochondria 70 of a 34-residue synthetic peptide corresponding to the presequence portion of 28 ornithine aminotransferase requires a 50 cytosolic factorz4. This factor was puri45 fied to homogeneity by affinity chroma30132 tography using the synthetic presequenceZ. It not only stimulated the W-ethylmaleimide-sensitive factor. import of mitochondrial precursor probPresequence-binding factor. teins but also increased their binding to CMitochondrial import stimulation factor. the mitochondrial surface, and was, therefore, called ‘targeting factor’. Both the molecular mass of the targeting factor (28 kDa) presequence and j3-galactosidase conjugated to a and its behaviour on ATP-agarose column chromaresin, which was then used to purify a cytosolic factography indicated that it was not hsp70 (Ref. 32). tor from rat liver cytosol. The factor was named mitoIn a second assay system, the in vitro import into chondrial import stimulation factor (MSF; Ref. 27). mitochondria of the purified precursor protein for MSF is a heterodimer of 32- and 30-kDa subunits, and ornithine carbamoyltransferase (OTC) was again it stimulates the mitochondrial import of all the prefound to be dependent upon the addition of a rabbit cursor proteins tested so far, even those synthesized reticulocyte lysate33. A protein factor with a molin the wheat-germ lysate system. These precursors ecular mass of 50 kDa was purified from the reticuloinclude those with cleavable presequences, such as cyte lysate, and named ‘presequence-binding factor’ pAd, pre-superoxide dismutase and a yeast Cox-IV(PBF). Binding of the factor to precursor proteins to presequence-porin fusion, but also precursors withform a soluble complex was dependent on the presout cleavable presequences, such as porin, an outer ence of a cleavable presequence34 and was inhibited mitochondrial membrane protein. Thus, it appears by synthetic peptides corresponding to known that MSF recognizes mitochondrial precursor promitochondrial presequencesz6. The ability of PBF to teins irrespective of the presence or absence of a stimulate import was not inhibited by treatment cleavable presequence and stimulates their import with NEM and did not depend on ATP hydrolysis. into mitochondria. Purified PBFstimulated the import of the precursors How does MSF stimulate the import of precursor of OTC, aspartate aminotransferase and malate proteins synthesized in a wheat-germ lysate? Such dehydrogenase in reticulocyte lysates that had been proteins are thought to be produced as aggregates depleted for PBF, and the PBF-dependent import of so, we suggested that MSF ‘depolymerizes’ and OTC precursor (pOTC) was stimulated further by unfolds the precursors. This ATP-dependent event the addition of hsp70. Taken together, these results may involve a conformational change of the precursor, as suggested by the observation that aggregated suggest that PBF may maintain the import competence of several mitochondrial precursor proteins in pAd is degraded almost completely by treatment with cooperation with hsp70 (Ref. 26). However, despite a low concentration of trypsin in the presence of MSF these encouraging results concerning PBF and, and ATP, but only undergoes limited proteolysis in indeed, targeting factor, no further characterizthe presence of MSF and AMP-PNP (Ref. 27). In addition, MSF forms a stable complex with ureaations have been reported. denatured precursors, keeping them in a conformation competent for importz9. The two activities of MSF - a conformational modulator of mitochondrial precursor proteins MSF, namely the import-stimulation activity and the Mitochondrial precursor proteins synthesized in ATP-dependent-unfolding activity are separate functions, the former activity being NEM-sensitive, but reticulocyte lysates are imported efficiently in vitro into isolated mitochondria, whereas those syn- the unfolding activity being NEM-insensitive27,29. thesized in the wheat-germ lysate system are Precursor-induced ATPase activity of MSF imported poorly or not at all, suggesting that the wheat-germ system lacks some factors necessary for MSF is an ATPase, and mitochondrial precursor proteins induce its ATPase activity, irrespective of mitochondrial protein import17. For example, the the presence or absence of the cleavable preadrenodoxin precursor (pAd) synthesized by the cellsequence29. However, mature forms of the mitofree wheat-germ lysate is not imported into isolated chondrial proteins, or proteins with extramitoliver mitochondria unless the cytosolic fraction from chondrial locations, do not induce such activity, the liver, reticulocyte or yeast is added to the import which suggests that MSF-ATPase is induced by the reaction. This general inability of proteins synmitochondrial-targeting sequence. Indeed, chemithesized in cell-free wheat-germ lysates to undergo cally synthesized peptides corresponding to the mitochondrial import was used in the design of an mitochondrial-targeting sequences bind to MSF and affinity matrix made of a fusion protein of Cox IV trends

in CELL BIOLOGY

(Vol.

6) March

1996

Refs 12-14 15 16-18 17 24,25,32 26,33,34 21-23 27-31,37

1

105

MSF-dependent pathway

MSF-independent pathway

hsp

OM In&membrane IM

FIGURE A model for precursor targeting Tom37-Tom70-dependent

to mitochondria. and MS&independent

Mitochondrial precursor

import stimulation targeting pathways

1

factor (MSF), are outlined.

The complex between MSF and adrenodoxin precursor (MS-pAd) docks onto Tom37-Tom70, MSF is then released and the precursor is transferred to Tom20-Tom22 (Ref. 30) probably through the acidic region at the cytoplasmic side of Tom20 and Tom22 (Refs 10, 11, 41 and 42). The presequence is then transferred to the intermembrane space through the translocation pore, an event that is probably initiated by the acidic domain of Tom22, which faces the intermembrane space41A3. Precursors that can maintain the unfolded conformations by themselves, or by the action of hsp70, bypass Tom37-Tom70 and are targeted directly to Tom20-Tom22. Note that the nomenclature of components of the protein transport machinery at mitochondrial membranes changed recently44.

induce its ATPase activityz8. The basic amino acid residues in the mitochondrial-targeting sequence are critical for recognition by MSF (Ref. 28). Furthermore, mitochondrial presequences can induce aggregation of unfolded proteins, such as reduced and carboxy-methylated a-lactoalbumin35, thus inducing a conformation that MSF might recognize better than depolymerized proteins. However, other experiments indicate that MSF-ATPase activity can be induced by additional structural elements within proteins destined for import into mitochondria: l Apocytochrome c does not contain a cleavable presequence and is imported into the intra-mitochondrial membrane space via a unique pathwaya6. Nevertheless, this protein induces MSF-ATPase activityzg. l Porin, the outer mitochondrial membrane protein, is synthesized as a mature form. The mitochondrialtargeting sequence of this protein has not been identified, but it too induces MSF-ATPase activity. l Import-competent, unfolded pAd only induces a low activity, whereas aggregated, import-incompetent pAd induces a higher degree of ATPase activityZ7Jg. These results suggest that MSF recognizes several features of the precursor proteins, including the conformational state of the mature region, in addition to the mitochondrial-targeting sequences. 106

The observation that aggregated pAd induces the ATPase activity of MSF led us to speculate that MSF catalyses unfolding of the aggregated precursors. Thus, MSF shares some properties with the molecular chaperones of the hsp70 family: MSF and hsp70-family proteins recognize aggregated proteins and unfold them in an ATP-dependent manner; they all complex with the unfolded proteins and stabilize the unfolded conformations; and all exhibit ATPase activity dependent on the presence of specific peptide sequences or aggregated proteins. The conspicuous difference between MSF and members of the hsp70 family is that MSF recognizes specifically the mitochondrial-targeting sequence and some additional structural elements of mitochondrial precursor proteins, whereas proteins from the hsp70 family bind much less specifically to those peptides enriched in hydrophobic amino acid residues. MSF- and hsp70-dependent pathways

targeting

Cytoplasmic ATP is required for the mitochondrial import of one group of precursor proteins37, suggesting the involvement of cytoplasmic chaperone proteins. Although both hsp70 and MSF are plausible candidates for this role, the MSF-dependent pathway appears to need ATP, whereas the hsp70 pathway does not. If a complex formed between hsp70 and pAd is incubated with isolated mitochondria in the absence of extramitochondrial ATP, hsp70 is released to the supernatant, and pAd is imported into the mitochondria31. AMP-PNP, which prevents the ATP-dependent dissociation of hsp70-pAd, does not inhibit the import of pAd. However, when an MSF-hsp70-pAd complex is incubated with mitochondria in the absence of extramitochondrial ATP, pAd import is arrested, hsp70 is released from the complex into the supernatant, and both MSF and pAd remain bound to the mitochondria. The import arrest can be relieved by the addition of ATP, which also causes the release of MSF from the membrane. These results indicate that the MSF-dependent import pathway requires extramitochondrial ATP to release MSF from the receptor on the outer membrane of the mitochondria, whereas the hsp70-dependent pathway does not (Fig. 1)31, and they suggest, therefore, that MSF is responsible largely for the ATP-dependent import of proteins into the mitochondria. The import of some proteins - for example, pAd into mitochondria is supported by both MSF and hsp70 to similar extents31. MSF- and hsp70-dependent import can, however, be distinguished, not only by ATP-dependency, but also by the degree of sensitivity to NEM: MSF-dependent import is inhibited by NEM, whereas hsp70-dependent import is not. trends

in CELL BIOLOGY

(Vol.

6) March

1996

NEM-insensitive, hsp70-dependent import becomes NEM-sensitive when increasing amounts of MSF are added31. Does this mean that both pathways are involved in mitochondrial import, and, if so, what is their relationship? Studies that used antibodies to label outer mitochondrial membrane proteins indicate that MSF- and hsp70-dependent precursor targeting occurs through distinct protein components on the membrane”‘. In the hsp70-dependent pathway, precursors that can assume an unfolded conformation, either by themselves or in the presence of hsp70, bind directly to the membrane receptor. For example, urea-denatured pAd can form a binary complex with hsp70. If this complex is incubated with the mitochondrial outer membrane, hsp70 dissociates from the complex in the absence of ATP hydrolysis, but pAd remains bound to the membrane. However, if urea-denatured pAd is incubated with hsp70 and MSF, a ternary complex is formed. Again, if the complex is incubated with the outer mitochondrial membrane in the absence of ATP, hsp70 is released, but MSF and pAd remain associated with the membrane. This result, in conjunction with the data showing that pretreatment of MSF with NEM abolishes its precursor-targeting functionz7J9, indicates that the precursors bind indirectly to the membrane through MSF and the membrane receptor in MSF-dependent precursor targeting”l. Interestingly, the binding of either hsp70-pAd or MSF-hsp70-pAd to the receptors in the membrane seems to cause a conformational change in the precursor and to induce the dissociation of hsp70 from the complex. Furthermore, the observation that a chemically synthesized, functional mitochondrial-targeting sequence, but not its mutated version, induces the binding of MSF to the outer membrane3r demonstrates that MSF only binds to the membrane in the presence of a correct targeting sequence. The early

steps of mitochondrial

protein

import

In Saccharomyces cerevisiae, four proteins in the outer mitochondrial membrane have been identified as components of the protein import receptor complex (Tom20, Tom22, Tom70, Tom37)38. These proteins form two subcomplexes, Tom20-Tom22 and Tom37-Tom70. The pad-induced ATPase activity of MSF is inhibited by the outer mitochondrial membrane, and the degree of inhibition correlates well with the degree of binding of MSF-pAd to the membrane. These findings have been used in combination with the results of studies using antibodies raised against the outer membrane components to determine which components interact with the MSF-precursor complex. The binding of urea-denatured pAd to mitochondria is inhibited by antibodies to Tom20 and Tom22 indicating that denatured proteins alone use the Tom20-Tom22 subcomplex as a receptor. However, further experiments indicate that the Tom37-Tom70 subcomplex acts as a receptor for the MSF-precursor complex. In a final set of experiments, import of MSF-pAd complex that had been prebound to mitochondria was inhibited by antibodies to Tom20 and Tom22. Taken together, these results indicate that the MSF-pAd complex docks onto trends

in CELL BIOLOGY

(Vol.

6) March

1996

Tom37-Tom70, MSF is then released and the precursor is transferred to Tom20-Tom22 and, subsequently, is translocated across the outer membrane (Fig. 1)30. Similar results have been obtained with rat liver mitochondria - an IgG raised against a 37-kDa protein of the outer membrane (OM37) inhibited MSF-dependent binding of pAd, but did not inhibit hsp70-dependent binding or import of pAd. By contrast, IgGs recognizing a rat homologue of Tom20 did not inhibit the binding of the MSF-pAd complex, but inhibited completely the subsequent import of pAd, as well as the hsp70-dependent binding of pAd (Ref. 31). Cytoplasmic requirements

chaperones

and energy

The foregoing evidence indicates that a mitochondrial precursor protein can be imported through either an NEM-sensitive, MSF- and Tom37-Tom70dependent pathway, or through an NEM-insensitive, MSF-independent pathway (Fig. 1). Both precursortargeting pathways function in parallel and their relative importance may be determined by the affinity of the precursor protein for MSF and hsp70. In the first pathway, the MSF-precursor complex docks onto Tom37-Tom70, MSF is released from the receptor in an ATP-dependent manner, and the precursors are transferred to Tom20-Tom22 before import into mitochondria. Precursors that are not recognized or are only recognized inefficiently by MSF, but are able to maintain the unfolded conformation either by themselves or by the action of hsp70, are targeted directly to Tom20-Tom22 and are transported across the outer membrane. This pathway does not require extramitochondrial ATP as the precursors either do not require hsp70 to keep in an unfolded conformation or, if they do, hsp70 dissociates spontaneously from the precursor upon interaction of the hsp70-precursor complex with Tom20-Tom22. Import studies with yeast mitochondria have shown that mitochondrial precursors can be classified into two groups35,36. The first group consists of hsp60, Cox IV, cytochrome c haem lyase and proteins fused with dihydrofolate reductase. These tend to assume a loosely folded conformation outside the mitochondria and do not require extramitochondrial ATP for their import. The second group includes the B-subunit of Fr-ATPase, a-subunit of mitochondrial processing peptidase, ADHIII, cytochrome c1 and adenine-nucleotide translocator. The import of the precursors of this group requires external ATP. The relationship between the requirement for extramitochondrial ATP and the relative importance of both MSF-dependent and -independent pathways remains to be elucidated for the aforementioned precursor proteins. Concluding

remarks

The function of MSF and hsp70 as described in this article has been examined and characterized only by the use of in vih-o systems. Although it is tempting to extrapolate directly from in vitro to in vivo studies, the function of MSF and other possible cytoplasmic factors must be studied now in vivo. MSF has been 107

identified as a member of the 14-3-3 family of proteins, members of which have been associated with many diverse intracellular functions, such as neurotransmitter biosynthesis, cell-cycle regulation, signal transduction and exocytosis39,40. Since the yeasts Schizosaccharomycespombe and Saccharomyces cerevisiaealso contain 14-3-3 proteins, genetic approaches with yeast may prove helpful in establishing the in tivo function of MSF in precursor targeting to mitochondria. References 1 VERNER, K. and SCHATZ, C. (1988) Science 241, 1307-l 313 2 BERNSTEIN, H. D., RAPOPORT, T. A. and WALTER, P. (1989) Cell 58, 1017-I 019 3 LITHGOW, T., HOJI, P. B. and HOOGENRAAD, N. J. (1993) FEBS fett. 329, l-4 4 WALTER, P. and JOHNSON, A. E. (1994) Annu. Rev. Cell Biol. 10, 87-l 19 5 PFANNER, N. and NEUPERT, W. (1990) Annu. Rev. Biochem. 59, 331-353 6 PFANNER, N., SijLLNER, T. and NEUPERT, W. (1991) Trends Biochem. Sci. 16, 63-67 7 SeLLNER, T., CRIFFITHS, C., PFALLER, R. and NEUPERT, W. (1989) Cell 59, 1061-l 070 8 SOLLNER, T., PFALLER, R., GRIFFITHS, G., PFANNER, N. and NEUPERT, W. (1990) Cell 62, 107-l 1.5 9 HINES, V., BRANDT, A., GRIFFITHS, G., HORSTMAN, H., BROTSCH, H. and SCHATZ, G. (1990) EMBOj. 9,3191-3200 10 RAMAGE, L., JUNNE, T., HAHNE, K., LITHGOW, T. and SCHATZ, G. (1993) EMBO]. 12,4115-4123 11 MOCZKO, M., EHMANN, B., GWRTNER, F., HONLINGER, A., SCHAFER, E. and PFANNER, N. (1994) 1. Biol. Chem. 269, 9045-9051 12 MIURA, S., MORI, M. and TATIBANA, M. (1983) I. Biol. Chem. 258,6671-6674 13 ARGAN, C., LUSTY, C. J. and SHORE, G. C. (1983) 1. Biol. Chem. 258,6667-6670 14 .PFANNER, N. and NEUPERT, W. (1987) /. Biol. Chem. 262, 7528-7536 15 OHTA, H. and SCHATZ, G. (1984) EMBOj. 3, 651-657 16 DESHAIES, R. J., KOCH, B. D., WERNER-WASHBURNE, M., CRAIG, A. E. and SHECKMAN, R. (1988) Nature 332, 800-805 17 MURAKAMI, H., PAIN, D. and BLOBEL, G. (1988) /. Cell Biol. 107,2051-2057

108

18

CHIRICO, W. J., WATERS, M. G. and BLOBEL, G. (1988) Nature 332,805-810 19 ZIMMERMANN, R., SAGSTETER, M., LEWIS, M. J. and PELHAM, H. R. B. (1988) EMBO 1. 7, 2875-2880 20 IMAMOTO, N. et al. (1992) 1. Cell Biol. 119, 1047-l 061 21 CAPLAN, A. J. and DOUGLAS, M. G. (1991)j. Ce//Bio/. 114, 609-621 22 CAPLAN, A. J., CYR, D. M. and DOUGLAS, M. G. (1992) Cell 71, 1143-1155 23 ATENCIO, D. P. and YAFFE, M. P. (1992) Mol. Cell. Biol. 12, 283-291 24 ONO, H. and TUBOI, S. (1988) 1. Biol. Chem. 263, 3188-3193 25 ONO, H. and TUBOI, S. (1990) Arch. Biochem. Biophys. 280, 299-304 26 MURAKAMI, K. and MORI, M. (1990) EMBO/. 9, 3201-3208 27 HACHIYA, N., ALAM, R., SAKASEGAWA, Y., SAKAGUCHI, M., MIHARA, K. and OMURA, T. (1993) EMBO]. 12,1579-l 586 28 KOMIYA, T., HACHIYA, N., SAKAGUCHI, M., OMURA, T. and MIHARA, K. (1994) I. Biol. Chem. 269,30893-30897 29 HACHIYA, N. eta/. (1994) EMBO/. 13,5146-5154 30 HACHIYA, N., MIHARA, K., SUDA, K., HORST, M., SCHATZ, G. and LITHGOW, T. (1995) Nature 376, 705-709 31 KOMIYA, T., SAKAGUCHI, M. and MIHARA, K. (1996) EMBO]. 15,399407 32 ONO, H. and TUBOI, 5. (1990) Arch. Biochem. Biophys. 277, 368-373 33 MURAKAMI, K., AMAYA, Y., TAKIGUCHI, M., EBINA, Y. and MORI, M. (1980) 1. Biol. Chem. 263, 18437-l 8442 34 MURAKAMI, K., TANASE, S., MORINO, Y. and MORI, M. (1992) 1. Biol. Chem. 257, 1311 Y-l 3122 35 ENDO, T., MITSUI, S. and ROISE, D. (1995) FEBS left. 359, 93-96 36 STUART, R. A., NICHOLSON, D. W. and NEUPERT, W. (1990) Cell 60, 31-43 37 WACHTER, C., SCHATZ, G. and CLICK, B. S. (1994) Mol. Biol. Cell 5, 465-474 38 LITHGOW, T., GLICK, B. S. and SCHATZ, G. (1995) Trends Biochem. Sci. 20,98-l 01 39 ALAM, R. et al. (1994) /. Biochem. 116,416425 40 AITKEN, A. et al. (1992) Trends Biochem. Sci. 17, 498-501 41 LITHGOW, T. et al. (1994) Proc. Not/ Acad. Sci. USA 9 1, 11973-I 1977 42 43 44

HONLINGER, A. et al. (1995) Mol. Cell. Biol. 15, 3382-3389 MAYER, A., NEUPERT, W. and LILL, R. (1995) Cell 80, 127-l PFANNER, N. et al. (1996) Trends Biochem. Sci. 21, 51-52

trends

in CELL BIOLOGY

(Vol.

6) March

37

1996